ES2234180T3 - SPECIFIC FIXING PROTEINS FOR THE TREATMENT OF CANINE ALLERGY. - Google Patents

Abstract

THE INVENTION REFERS TO UNION PROTEINS WHICH SPECIFICALLY JOIN AN ISOLATED AND PURIFIED PEPTIDE THAT INCLUDES A LEUKIN SITUATED TO A DISTANCE OF TWO PEPTIDIC LINKS OF A TYROSINE-ARGININE COUPLE. OTHER PROTEIN OF UNION OF THE INVENTION INCLUDE THOSE THAT SPECIFICALLY JOIN AN ISOLATED AND PURIFIED PEPTIDE WITH THE CISTEINE SEQUENCE - HOLLOW - PROLIN - HISTIDINE - HOLLOW - HOLLOW - HOLLOW - CYSTEINE - HISCINE - HISTORINE - PROTEIN - HISCINE - HISTORINE - HISCINE - PROTEIN - HISTORINE - HISCINE - HISTORINE - PROTEIN - HISCINE - HISTORINE - PROTEIN - HISCINE - PROTEIN PROLINA HUECO - HUECO - CISTEINA.

Description

Specific binding proteins for Canine allergy treatment.

Field of the invention

This invention relates to peptides. More in specifically, the invention relates to compositions for the administration to dogs, which actively provide immunity to the immunoglobulin E molecules of the dog.

Prior art

It is estimated that up to 30% of total dogs suffer allergies or skin disorders related to allergies. In It is estimated that allergic dermatitis affects between 3 and 15% of the entire canine population. Given the predominance of Allergies in dogs, there is a need to develop methods and compositions to correctly diagnose and treat dog allergies

The substances that cause with greater Probability an allergic reaction vary from one species to another. The Common allergens of dogs include fleas, pollen, mold and dust. Flea allergy is believed to be allergy most frequent canine. Flea saliva is a typical allergen and a single flea bite is enough to cause an itch considerable. Another additional form of canine allergy is called atopy Atopy is a pathological state in which the dog is allergic to certain inhaled substances, for example pollen, the mold and microscopic mites found in the dust domestic.

Antibody molecules play a role in allergic manifestations. In mammals, the molecules of antibodies are classified into various isotypes, called IgA, IgD, IgE, IgG and IgM. The antibody molecules consist of Long and short chain components. The long chains of the molecules of a given isotype have large regions of amino acid sequence homology and, conversely, have regions that differentiate them from antibodies belonging to other isotypes The common regions of the long chains provide members of each isotype with common capabilities of fixation on certain cell surface receptors or on other macromolecules, for example complementary. These regions of long chain are therefore used to activate the functions particular immune effectors. Consequently, the separation of antibody molecules in isotypes serve to separate the antibodies according to a set of effector functions that They usually activate.

In human persons and dogs, the Immunoglobulin E (then IgE) is involved in allergy. The IgE is thus the type of antibody that is believed to act as a important mediator of allergic responses, including immediate hypersensitivity of type I.

IgE molecules are fixed on mast cells and basophils This fixation takes place when the Fc region of the IgE molecule is fixed on mast cell Fc receptors. When such fixed IgE antibodies bind to an allergen, then the allergen binds and crosslinks various IgE antibodies on the surface mobile. This binding and crosslinking causes (medium) reactions of immediate hypersensitivity of type I and causes the release of histamines and other molecules that produce the symptoms associated with allergy

Monoclonal antibodies have been identified that have varying degrees of sensitivity to canine IgE and IgG (DeBoer et al., Immunology and Immunopathology 37 , 183-199, 1993). DeBoer et al. they have identified several monoclonal antibodies that have cross-reactivity with IgG and IgE (see eg DeBoer et al., table 4 and annexed text). DeBoer et al. They have identified three monoclonal antibodies (A5, D9 and B3) that have some affinity with canine IgE. Of the monoclonal antibodies identified by DeBoer et al., The D9 antibody appears to be the one with the highest degree of neutralization of the Prausnitz-Kustner reactivity of atopic canine serum. In the context of canine allergy, DeBoer et al. propose the use of their monoclonal antibodies (MAb) in the antigen-specific IgE ELISA and to quantify canine IgE. They also propose using their Mab for the immunostaining of the Western Blot assay to assess the molecular specificity of IgE antibodies, as well as for " in vitro " studies of mast cell degranulation.

In humans, the level of total serum IgE is a diagnosis of allergic disease. Several studies have been carried out to explore the possibility that the level of serum IgE can also be used as a diagnosis in dogs (Hill and DeBoer, Am. J. Yet. Res. 55 (7), 944-48, July 1994). Publications following the DeBoer article describe the use of a monoclonal antibody, called D9, in the ELISA assay that has the following configuration: D9 is fixed on a substrate, antibodies are captured by D9 and then D9 provided with a marker to signal the captured antibody. The Hill and DeBoer ELISA tests are applied to establish the total amount of IgE in canine serum, in an effort to diagnose canine allergy. Unlike humans, the amount of IgE determined as existing in the canine circulation is not at all useful for the diagnosis of allergy in dogs (see, eg, the summary and discussion sections of Hill and DeBoer). This finding contrasts greatly with the situation in human immunology.

This divergent diagnostic result, based in IgE levels in humans versus levels existing in dogs, points to the difficulty that must be overcome any attempt to correlate data between animals belonging to two different genres. This difficulty is aggravated. also because dogs can be allergic to sets of antigens different from those that cause allergy in humans. In addition, in cases where dogs and humans seem to be allergic to the same allergenic extracts, the studies conducted by doctors Esch and Greer de los Greer Laboratories indicate that the specific allergens of an extract allergen that causes canine disease are not necessarily the same allergens that cause disease in humans. By For example, it is known that the immunodominant components of Dust mite extracts are different in dogs and in humans.

Genomic sequences encoding the long chain constant region of murine and human IgE are known (see for example Ishida et al., "The Nucleotide Sequence of the Mouse Immunoglobulin E Gene: Comparison with the Human Epsilon Gene Sequence", EMBO Journal 1 , 1117-1123, 1982). The comparison of the human and murine genes indicates that they have a 60% homology within the exons and a 45-50% homology between the introns, with various insertions and deletions.

Patel et al. publish the predictable nucleotide and amino acid sequence of exons 1-4 of the canine IgE long chain constant region in an article entitled "Sequence of the Dog Immunoglobulin Alpha and Epsilon Constant Region Genes", Immunogenetics 41 , 282-286 (March 22, 1995). The complete sequence of the long chain constant region of the canine IgE, with membrane-fixed portions encoded by exons 5 and 6, is published in pending applications serial number 08/800 698, registered on February 14, 1997; 09/146 400, registered on September 3, 1998; and 09/146 617, registered on September 3, 1998.

It is believed that IgE is involved in the symptoms allergic, so it is desirable to decrease IgE levels as mechanism to relieve allergic symptoms. However, the a patient's own IgE molecules are autoproteins and the Immune responses to these proteins are usually suppressed. The suppression of immune responses to autoproteins is that is, a tolerance to autoantigens is supposed to take place In a large number of ways.

The current hypothesis of T cell suppression targeting autoantigens implies an induction of "deletion clone "of such T cells in the thymus, in which they are removed T cell receptors that can recognize self-peptides in association with MHC molecules and only the expansion of those that recognize foreign peptides and MHC molecules. further there may also be suppressor T cells that prevent induction of immune responses against autoproteins.

Unlike the situation of T cells, it believes that there are many B cells that are expressed in receptors (it is say, surface immunoglobulin) of autoproteins and that the reason why these cells do not produce antibodies against autoproteins is that T cells are normally missing required for the presentation of B cell antigen.

A B cell that recognizes epitopes (sites of antigen binding) of the IgE antibodies characteristic of a patient is able to generate antibodies, in general IgG, directed against this autoantigen, that is, IgE. Thus, the existence of such B cells constitutes a unique opportunity to induce the production of autoantibody responses. Follow there being a need for such antibodies in order to treat a allergic disease

The hypothesis regarding the "presentation of antigen "means: recognition of the antigen by a B cell surface immunoglobulin, internalization and to the processing of this antigen, the association of derived peptides of the antigen with MHC molecules, expressed on the surface of the B cells and then antigen peptide recognition associated and MHC molecules by a specific T cell. The interaction between T cells and B cells then leads to a signal transduction in both cells and to the synthesis and production of soluble cytokines that eventually result in Antibody production by B cells.

Therefore, in most circumstances, only when an antigen is foreign does a response occur immune; in the other cases, the internalization and processing of the autoproteins will usually lead to the presentation of self-peptide-MHC complexes to T cells and it this will translate into autoimmune antibodies.

Therefore, in order to induce a response from antibody to a self-peptide, for example IgE, the system immune has to be manipulated so that it allows a B cell Self-reactive becomes an antibody-secreting B cell. There is still a need to manipulate the immune system of this way, particularly in the context of diseases allergic

There are, in general, various known attempts to generate peptide antigen antibodies. For example, multiple antigenic peptides (MAP), introduced by Dr. James Tam (Tam, JP, Proc. Natl. Acad. Sci. USA 85 , 5409-5413, 1998) have various advantages in inducing antiseptic antibodies. The MAP strategy is an alternative attempt to the conventional technique of conjugating a peptide antigen with a protein support. One of the primary limitations of the use of protein supports is the great weight of the support with respect to the peptide antigen attached to it. This relative disparity in sizes can result in a low proportion of antiseptic antibodies if compared to anti-support antibodies. MAPs have, for example, 4 or 8 peptide arms that start from a central lysine skeleton, as shown in Figure 1 AB. The peptide antigen is conjugated with each of the arms.

Therefore, in a MAP system there is a much higher proportion of the support molecule's antigen than in traditional protein conjugation. This design maximizes the concentration of the antigen for the specific immunogenic response. Moreover, it has been observed that the central lysine nucleus of the MAP peptide is not immunogenic (Tam, JP, Proc. Natl. Acad. Sci. USA 85 , 5409-5413, 1988; Posnett, DN, McGrath, H. and Tam , JP, J. Biol. Chem. 263 , 1719-1725, 1988). Therefore, antibodies induced against MAP peptides are a direct response against the antigen. Accurate knowledge of the chemical composition, structure and amount of peptide before immunization is possible by direct synthesis of the antigen on the lysine branching nucleus. In addition, since MAP's attempt eliminates the need to conjugate peptides with support proteins, which can alter antigenic determinants, chemical ambiguity is also eliminated. It is believed, therefore, that MAP induce antibody responses of high purity, greater avidity, precise chemical definition and greater safety.

Fmoc MAP resins (supplied by Applied Biosystems, Foster City, California) are compatible resins with Fmoc, connected to a small central structure of remains branching lysine. The central structure contains several levels of lysine residues fixed to the previous lysine in both groups amino N-? and N-?, as depicted in figure 1A-B.

MAP peptides used in the experimental vaccine design have shown high concentrations of antiseptic antibodies, which recognize the native protein (Tam, JP, Proc. Natl. Acad. Sci. USA 85 , 5409-5413, 1988; Posnett, DN, McGrath, H. and Tam, JP, J. Biol. Chem. 263 , 1719-1725, 1988; Auriault, C., Wolowczuk, I., Gras-Masse, H., Maguerite, M., Boulanger, D., Capron, A. and Tartar, A., Peptide Res. 4 , 6-11, 1991). In addition, greater sensitivity and reliability of antibody-antigen interactions in solid phase immunoassays with MAP peptides has been observed, due to a greater capacity and avidity of coating (Tam, JP and Zavala, F., J. Immunol. Meth. 124 , 53-61, 1989).

An additional strategy, which is known to It is useful for generating peptide antigen antibodies, it consists of place multiple copies of peptides on the surface of particles of plant viruses. An example of this is EPICOAT ™ technology (Axis Genetics plc, Cambridge, England). EPICOAT ™ technology It is based on chimeric virus particle technology (CVP) which uses the recombinant genetic modification of viruses the plants.

EPICOAT ™ technology involves the insertion of a small portion of a foreign protein (a peptide) in a virus of a plant, such that they are generated on the surface of the virus particle multiple copies of the peptide. The technology EPICOAT ™ is currently based on the bean mosaic virus of black eye (cowpea) (CPMV), a plant virus that infects the plant of the bean, plant is also known as "black-eyed bean". The CPMV particle without modify is an icosahedron and has a diameter of approximately twenty eight nanometers (nm). CPMV particles are made up of two proteins, called large coat proteins and small. Studies indicate that within the protein of small coating there is a site that allows the presentation of a foreign peptide in a prominent position on the surface of the virus, whereby in each virus particle can occur up to sixty copies of a specific peptide.

With EPICOAT ™ technology, DNA copies of the plant virus genetic material are used. A tiny amount of the DNA encoding the virus protein, including the inserted foreign peptide, along with an abrasive powder is applied to the leaves of a young black-eyed bean plant. After rubbing gently, the DNA is incorporated into the leaves and uses the plant's own cellular mechanisms to start the generation of the virus's functional particles. The virus replicates inside the inoculated leaves and spreads through the plant in
increase.

After two or three weeks, foliar material is collected that contains considerable amounts of the virus. Chimeric virus (CVP) particles are isolated by centrifugation and selective precipitation of homogenized plant material. 1 to 2 grams of CVP are obtained per kilogram of fresh foliar material. Black-eyed bean plants are grown in abundance in controlled environments, resulting in the production of an abundant amount of
CVP

It has been published that peptides of a size of up to thirty-six amino acids have been incorporated into the CVP. The resulting particles are extraordinarily robust, with a thermal inactivation point of 65 ° C. It is observed that CVPs resist an acidic pH as well as protein degradation enzymes. It has been published that expressed peptides are capable of producing specific immune responses in animals. The hypothesis that the superficial presentation of peptides can improve recognition by the host immune system and can provide a developmental pathway to recombinant subunit vaccines and immunotherapeutic agents has been launched.
Peutics

DeBoer et al., 1993 (Veterinary Immunology and Immunopathology 37 , 183-199, 1993) have prepared a series of murine hybridoma cell lines that produce monoclonal antibodies specific to canine immunoglobulins E and G. The ability of these monoclonal antibodies is checked for inducing a reversible cutaneous anaphylaxis reaction on the dog's skin, to neutralize the Prausnitz-Küstner reactivity of atopic dog serum, to serve as a ligand in immunoaffinity chromatography and to fix on IgE and other Ig subgroups in various systems ELISA Some of these monoclonal antibodies produced are found to be specific to canine IgE.

Konieczny et al., 1997 (Immunology 92 , 577-586, 1997) publish the isolation of cDNA encoding the main allergenic proteins and presenting their nucleotide sequences and deduced amino acid sequences. Both proteins (VEG and MUP proteins) are members of the small ligand binding protein lipocalin family. Recombinant forms of these proteins are produced and their reactivity with immunoglobulin E is verified. In addition, both recombinant proteins are able to bind and cross-link IgE and produce histamine released " in vitro " by peripheral blood leukocytes.

Burt et al., 1987 (Molecular Immunology, vol. 24, No. 4, pp. 379-389, 1987) describe the production of polyclonal antisera with predetermined specificities for a fan of rat IgE epitopes by immunization of rabbit KLH conjugates of five different synthetic peptides, which they present different sequences in the CH3 and CH4 domains of the IgE of rat. Antiseptic sera are analyzed in functional assays. designed to investigate the mode of interaction between the IgE of rat and its receptor in murine mast cells. Each serum of anti-peptide is able to inhibit the fixation of the IgE to mast cells and also capable of initiating the secretion of histamine from cells sensitized with rat IgE in a mode induced by "anti-IgE".

Presta et al., 1993 (Journal of Immunology, vol. 151, 2623-2632, No. 5, 1993) describe the humanization of a murine antibody that is able to block the binding of IgE to its high affinity receptor, but that is not fixed on the IgE after it has been fixed on the receptor, because this would trigger histamine production. Be evaluate variants of humanized antibody to test the importance of the remains of the structure in fixing antibodies and to determine what remains loaded from the CDRs interact with IgE. It has been found that only require five changes in the remains of the human structure to provide binding comparable to that of the murine antibody original.

Description of the invention

A specific binding protein is described that specifically binds on exon 3 of the native canine IgE free or fixed on B cells and that does not bind on exon 3 of the IgE when the IgE is bound to the receptor in a mast cell The surface bound IgE can be the IgE expressed on the surface of a B cell
canine

A specific binding protein that specifically binds an isolated and purified peptide containing a leucine positioned two peptide bonds beyond the tyrosine-arginine pair is described; eg, SEQ ID NO: 1 leucine-white-white-tyrosine-arginine, SEQ ID NO: 2 tyrosine-arginine-white-white-leucine or SEQ ID NO: 3 leucine-white-white-tyrosine -arginine-white-white-leucine. The peptide may contain from 5 to 71 amino-
acids

An antibody is described which is fixed on a epitope defined and that is cultured to obtain an isolated peptide and purified containing an amino acid sequence or a variant Conservative of it, which contains: SEA ID NO: 4 Thr-Leu-Leu-Glu-Tyr-Arg-Met; a recombinant binding molecule that is fixed is also published specifically about the defined epitope set by the antibody.

An antibody is described which is fixed on a epitope defined and that is cultured to obtain an isolated peptide and purified containing an amino acid sequence or a variant Conservative of it, which contains: SEA ID NO: 5 Gly-Met-Asn-Leu-Thr-Trp-Tyr-Arg-Glu-Ser-Lys; a recombinant binding molecule that is fixed is also published specifically about the defined epitope set by the antibody.

A specific binding protein that is cultured to obtain a multiple antigenic peptide containing multiple copies of an isolated and purified peptide containing a leucine moiety positioned two peptide bonds beyond the tyrosine-arginine pair is also published; the specific binding protein is specifically fixed on a defined epitope. The isolated and purified peptide may have 5 to 71 amino acids. A recombinant binding molecule is also published that specifically binds to a defined epitope bound by the protein of
fixation.

A specific binding protein is published which binds specifically on an isolated and purified peptide which contains a cysteine group positioned two peptide bonds beyond the proline-histidine pair, positioned three peptide bonds beyond the cysteine rest. The peptide can have the form: SEQ ID NO: 6 cysteine-white-white-proline-histidine-white-white-white-cysteine. Cysteines can form a covalent bond through a reduction reaction, peptide cyclization and incorporation Cystine The peptide may contain 9 to 71 amino acids.

An antibody that binds to an epitope is published defined and grown to obtain an isolated peptide and purified that has the amino acid sequence, or a variant Conservative of it, consisting of: SEQ ID NO: 7 serine-valine-threonine-leucine-cysteine-proline-asparagine-proline-histidine-isoleucine-proline-methionine-cysteine-glycine-glycine-glycine. Cysteines can form a covalent bond through a reduction reaction, peptide cyclization and incorporation Cystine A fixation molecule is further described. recombinant that binds specifically to the defined epitope fixed by the antibody.

An antibody that binds to an epitope is published defined and grown to obtain an isolated peptide and purified that has the amino acid sequence, or a variant Conservative of it, consisting of: SEQ ID NO: 8 serine-alanine-cysteine-proline-asparagine-proline-histidine-asparagine-proline-tyrosine-cysteine-glycine-glycine-glycine. Cysteines can form a covalent bond through a reduction reaction, the cyclization of the peptide by changing the amino acid by cystine. A fixation molecule is further described. recombinant that binds specifically to the defined epitope fixed by the antibody.

A specific binding protein is published which specifically binds to an isolated and purified peptide that it contains a cysteine residue positioned a peptide bond beyond of the proline-histidine pair, positioned a bond peptide beyond a proline residue, positioned two links peptides beyond a cysteine residue; the peptide can have the form of SEQ ID NO: 9 cysteine-white-proline-histidine-white-proline-white-white-cysteine. Cysteines can form a covalent bond through a reduction reaction, peptide cyclization and incorporation Cystine The peptide may contain 9 to 71 amino acids.

An antibody that binds to an epitope is published defined and grown to obtain an isolated peptide and purified that has the amino acid sequence, or a variant Conservative of it, consisting of: SEQ ID NO: 10 serine-alanine-cysteine-histidine-proline-histidine-leucine-proline-lysine-serine-cysteine-glycine-glycine-glycine. Cysteines can form a covalent bond through a reduction reaction, peptide cyclization and incorporation Cystine A fixation molecule is further described. recombinant that binds specifically to the defined epitope fixed by the antibody.

Specific binding proteins according to the invention may be antibodies, both polyclonal and monoclonal For example, the monoclonal antibody may be the 8H.8 or 15A.2. The antibody according to the invention can fix a defined epitope; recombinant molecules of fixation that are fixed specifically on the defined epitope bound by an antibody according to the invention.

The use of a protein is also published specific fixation according to the invention for the manufacture of a pharmaceutical composition intended for the treatment and prophylaxis of canine allergy In a preferred use, this pharmaceutical composition It also contains a diluent. It also publishes the use of a specific binding protein for the manufacture of a kit pharmaceutical composition, said kit contains the first dose of said specific binding protein and a second dose of said specific binding protein.

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Definitions Amino acids

one

CDNA clone: a DNA duplex sequence that represents an RNA, transported by a cloning vector.

Cloning: the selection and propagation of a individual species of DNA.

Cloning vector: a plasmid, phage DNA or other DNA sequence, capable of replicating in a host cell and capable of transporting the DNA sequence added exogenously for purposes amplification or expression of the added DNA sequence.

Codon: a nucleotide triplet that represents an amino acid or termination signal.

Conservative variants: the variants conservative nucleotide sequences include the nucleotide substitutions that do not produce sequence changes of amino acids encoded by said nucleotides, as well as the nucleotide substitutions that produce substitutions amino acid preservatives, eg amino acid substitutions that do not substantially affect the character of the polypeptides translated from said nucleotides. For example, the character of a IgE-derived peptide is not substantially affected if the substitutions do not exclude specific binding of the peptide on the canine IgE receptor or other IgE binding ligands canine

The conservative sequence variants of amino acids include substitutions or deletions of amino acids that do not substantially affect the character of polypeptide of the variant with respect to the starting peptide. By example, the polypeptide character is not affected substantially if substitutions or deletions do not exclude specific fixation of the variant peptide on a site corresponding specific binding of the starting peptide. He "mimotopo" refers to a conservative variant of a amino acid sequence, with respect to which a antibody specificity. The mimotope consists of a variant of the epitope of the starting peptide, so that it is able to fix antibodies that may have a cross reaction with the epitope original.

DNA sequence: a linear series of nucleotides connected to each other by phosphodiester bonds between the carbons 3 'and 5' of adjacent pentoses.

Expression: the process that a gene undergoes structural to produce a polypeptide. It is a combination of transcription and translation.

Expression control sequence: a sequence of nucleotide DNA that controls and regulates gene expression structural when operatively binds to these genes.

Exon: a contiguous region of the DNA that encodes a portion of a polypeptide. When an exon is mentioned either, eg "exon 6 DNA sequence", the complete exon or a portion thereof.

Genome: the complete DNA of a substance. It includes among others the structural genes that encode the polypeptides of the substance as well as the operator fixation sequences, promoter and ribosome and interaction sequences, for example the Shine-Dalgarno sequences.

Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar (pentose) moiety, a phosphate and a base nitrogen heterocyclic. The four bases of DNA are adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). The four bases of the RNA are A, G, C and the uracil ("U"). A and G are purines and C, T and U are pyrimidines

Phage or bacteriophage: bacterial virus, many of they include DNA sequences encapsulated in a lining or protein coating ("capsid").

Plasmid: an extrachromosomal circular DNA self-replicating autonomous.

Polymerase chain reaction (PCR): a method amplification of an existing target DNA sequence in a DNA sequence mixing, using oligonucleotide primers that flank the target DNA sequence during repeated cycles of DNA synthesis of the target DNA sequence.

Polypeptide: a linear series of amino acids connected to each other through peptide links between the groups α-amino and carboxyl of adjacent amino acids.

Reading frame: codon grouping during the translation of mRNA into amino acid sequences. By For example, the GCTGGTGTAAG sequence can be translated into three frames of reading or phases, each of which provides a sequence other than amino acids:

GCT GGT TGT AAG-Ala-Gly-Cys-Lys

G CTG GTT GTA AG-Leu-Val-Val

GC TGG TTG TAA A-Trp-Leu- (STOP).

Recombinant DNA molecule: a sequence of Hybrid DNA that contains at least two nucleotide sequences, the first sequence is usually not found in nature along with the second.

Specific fixation: the fixation of a substance with another for a fixation affinity greater than the fixation of background. Two substances that have specific binding are called specific fixing torque. An antibody and an antigen are a example of specific fixing torque.

Specific binding molecule: a molecule that presents a specific fixation with its corresponding partner of fixation to form a specific fixation pair. As it use in this description, this definition of binding molecule specific comprises monoclonal and polyclonal antibodies, the antigen binding fragments of said antibodies, the hybrid antibodies, single chain antibodies and recombinant molecules capable of specifically binding on a ligand.

Structural gene: a DNA sequence that encodes by its template or messenger RNA ("mRNA") a amino acid sequence characteristic of a polypeptide specific.

Transcription: RNA synthesis on a template of DNA

Translation: synthesis of peptides on a template of mRNA.

Description of the figures

A structure of nucleus of a four-arm MAP protein, that is, a protein 4-MAP; Figure 1B shows the structure core of an eight-arm MAP protein, that is, a protein 8-MAP.

Figure 2 shows the data comparatives of the fixing characteristics of various monoclonal antibodies on surface-bound IgE (e.g., similar to IgE expressed on the surface of B cells); he IgE receptor (eg similar to the Fc receptor on mast cells); and on a combination of IgE when set by the IgE receptor. Each component tested is therefore immobilized on a solid surface.

Figure 3 shows the capacity of exon recombinant 3 to inhibit binding of conjugated antibody 8H.8 or of conjugated antibody 15A.2 on a solid phase of IgE. The 8H.8 data is represented by dotted bar charts and 15A.2 data is represented by bar graphs black continuous.

Figure 4 shows the capacity of a Purified soluble canine IgE to inhibit the fixation of a 8H.8 monoclonal antibody (2.5 µg / ml) or an antibody monoclonal 15A.2 (2.5 µg / ml) on a solid phase of IgE canine The 8H.8 data is represented by graphs of dotted bars; the data of 15A.2 is represented by continuous black bar graphs.

The capacity of the monoclonal antibody 15A.2 or monoclonal antibody 8H.8 of inhibit the binding of IgE on the recombinant IgE receptor immobilized on a solid phase, for example the one detected by a monoclonal antibody D9.

The sequences of amino acids from the phage display library PhDc7c of the New England Biolabs company, which is fixed by the antibody monoclonal 15A.2. These sequences are aligned with the protein sequence of canine IgE.

Figure 7 shows the alignment of the 15A.2 fixation region of seven different mammals: dog, Man, green monkey, cat, pig, mouse and horse.

Figure 8 shows the phage capacity that presents mimotopo 15A.2 peptides to inhibit that canine IgE fix monoclonal antibody 15A.2 on the solid phase.

Figure 9 shows the capacity of a specific mimotopo 15A.2 peptide sequence, fixed on the phase solid, to fix the mimotopo 15A.2 monoclonal antibody.

In figures 10A and 10B the ability of the 15A.2 mimotopo peptide to prevent the antibody monoclonal 15A.2 fix the canine IgE on the solid phase.

The capacity of the monoclonal antibody 8H.8 and monoclonal antibody 15A.2 of look at the purified canine IgE, immobilized on the phase solid.

Figure 12 shows the data of a evaluation study of the capacity of conjugated 15A.2 antibody with a rest signal to look at the solid phases that have several peptides immobilized thereon.

Figure 13 shows the data of a monoclonal antibody capacity assessment study 14K2, known to bind to exon 4 of canine IgE, to bind on several peptides immobilized on the solid phase.

Figure 14 shows the data of a monoclonal antibody capacity assessment study 15A.2, known for fixing on exon 3 of canine IgE, for fix on several peptides immobilized on the phase solid.

Figure 15 shows the data of studies comparing the binding of monoclonal antibody 8H.8 or of a monoclonal antibody obtained from recombinant IgE (Re-hu IgE), to look at a solid phase that has the E3a.5 peptide or a substituted E3a.5 peptide (called S3a.5) immobilized on it.

Figure 16 shows the data of a evaluation study of the capacity of the conjugated 8H.8 antibody with a signal rest to be fixed on solid phases that have various peptides immobilized on them.

Figure 17 shows the capacity of polyclonal anti-human IgE antibodies to fix on several peptides immobilized on the phase solid.

Ways to implement the invention Proposed mechanism of action for monoclonal antibodies anti-IgE to cause persistent depletion of serum IgE

A hypothesis according to the present invention consists of the monoclonal antibodies anti-IgE affect IgE levels by setting directly to the resident circulating IgE, after which it Remove the fixed complex from the circulation.

There is also the hypothesis that antibodies Anti-IgE monoclonal affect IgE levels interfering with the production of new IgE in B cells. memory B cells, cells that after interacting with the antigen and T cells becomes producing cells of antibodies (ie, "IgE memory B cells"), intervene in the replacement of serum IgE and these cells contain the IgE as its surface cell receptors. Of this mode it is possible for monoclonal antibodies anti-IgE are fixed on IgE in these cells producers of antibodies and interfere with their functioning, by example by restrictive regulation or by one or several mechanisms that They lead to cell destruction. Two general routes have been proposed to explain that the binding of a monoclonal antibody to a memory B cell can interfere in the production of the IgE

First, the activation of a B cell of memory may also require the intervention of a factor of IgE fixation on surface, called CD23. The attachement of certain monoclonal antibodies on cell surface IgE it can exclude the fixation of the CD23 and, in this way, can lead to the inability to mount a response to the IgE.

Second, the antibody production of IgE by a memory B cell can be reduced or removed by fixing a monoclonal antibody that is directed directly against or blocking the receiver of the IgE molecule, which is located on the surface of cell B of memory.

However, in the absence of an antibody monoclonal leading to the elimination or inactivation of a cell IgE memory B, would be expected that memory B cells of IgE would be replenished, leading to an eventual replacement of the Circulating IgE.

Serum IgE levels are elevated in Patients suffering from an allergic disease. As described here, when antibodies that are fixed are generated specifically on a species IgE and these are administered antibodies to a patient who is a member of that species ("passive immunization"), lower levels of IgE in the patient serum As the expert will appreciate in the matter, the specific binding proteins according to the invention are administered in pharmacologically acceptable doses and may administered in suitable biocompatible diluents. They describe also herein a method and related compositions which serve to induce a response to a self-peptide, for example an IgE molecule, for this the immune system is manipulated so that allows a self-reactive B cell to become a antibody-secreting B cell ("active immunization").

Receptors are present in mast cells that set the IgE in circulation. When the IgE that set the mast cell receptors, IgE molecules can remain fixed by cross union. After the cross union of these IgE molecules, the mast cell is induced to release histamine. The histamine is an agent that induces the manifestation of symptoms allergic

In general, the cross union takes place by binding of IgE molecules to an antigen, eg an allergen. However, a monoclonal antibody that binds IgE could serve as a means for cross-linking of IgE that has been remained attached to the surface of the mast cells.

In the case of allergic individuals, if they exist anti-IgE antibodies in circulation, it's easy understand that the fixation of such molecules auto-anti-IgE to the IgE of the mast cells can serve to cross-link the molecules of IgE on such cells and exacerbate a continuous allergic response even in the absence of allergen. This event is clearly undesirable in the context of allergy treatment. A antibody that performs this function will therefore not be preferred for use as a therapeutic agent according to the invention. By consequently, an antibody that effects such cross-linking is disadvantageous and not preferred according to the invention.

There are two strategies to produce antibodies monoclonal targets that target IgE, but do not produce cross-linking of IgE molecules, which have been fixed to a mast cell. The first is to produce antibodies monoclonal targeting an epitope of an IgE molecule that It is only accessible when the IgE is in circulation. Dice that the monoclonal antibody can only be fixed on IgE when it is in circulation, it will not be able to notice the IgE that remained attached to the surface of a mast cell.

An alternative strategy to produce anti-IgE antibodies has been proposed by Stanworth et al., Eg US-5 601 821, published on February 11, 1997. Stanworth describes antibodies that cause cross-linking of IgE on mast cells, but so that they do not induce histamine release. Stanworth has described an epitope of the human IgE molecule, which has to be available or accessible after the IgE molecules are have been joined by cross-union on the surface of a mast cell, in order to release mast cell histamine. Of this mode, Stanworth describes an antibody that binds to an epitope Concrete of this type. Therefore, the antibodies monoclonal targeting this epitope of IgE will serve for the cross-binding of the IgE, but will not allow the mast cell to release histamine

The strategy followed according to the present invention is the development of antibodies, either " ex vivo " monoclonal antibodies, either " in vivo " anti-autoantibodies, directed against epitopes that are accessible in circulating IgE, but which are not accessible when the IgE is fixed to a mast cell.

Passive immunity Monoclonal antibodies 15A.2 and 8H.8

Monoclonal antibody 15A.2 is fixed on the Canine IgE Monoclonal antibody 15A.2 is derived by immunization of mice with affinity purified canine IgE. He Epitope set by 15A.2 is conformational, not linear. Such as indicate the data represented in figure 2, 15A.2 is not fixed on the IgE receptor; neither does it fix on the IgE when This is attached to the receiver. However, 15A.2 has affinity with respect to free IgE. It is fixed on fusion proteins of recombinant canine exon 3 in an immunosorbent assay linked to enzyme (ELISA) or in a Western Blot assay, but not over others recombinant canine IgE fusion proteins.

Antibody characterization assays monoclonal 15A.2 indicate that 15A.2 is not fixed on the IgE that already is bound to the mast cell IgE receptor. That is why it puts manifest that access to the epitope set by the 15A.2 due to the binding of IgE on the Fc receptor of the mast cells Therefore, 15A.2 does not produce cross-linking of IgE fixed to mast cells. This finding is demonstrated with the fixation studies on a recombinant receptor described at the moment.

The 8H.8 monoclonal antibody is also fixed about canine IgE. The 8H.8 monoclonal antibody is derived by immunization of mice with a reduced version of exon 3 of the Canine IgE molecule, called exon 3a. Exon 3a contains the 71 C-terminated amino acids of full-length exon 3. Previous studies (whose data are not presented here) indicate that immunization of mice with exon 3 in length complete does not generate antibodies with specificity, such as which has been found lately in the 8H.8 antibody.

Antibody characterization assays monoclonal 8H.8 indicate that, unlike antibodies monoclonal derivatives by immunization with all others recombinant IgE sequences, 8H.8 would also be fixed on the Native canine IgE. Also, like 15A.2 and the mode represented in figure 2, it has been found that 8H.8 is not fixed on the IgE that is already bound to the mast cell IgE receptor. Therefore, it seems that access to the epitope is also prevented set by 8H.8 due to the fixation of IgE on the receptor Fc of mast cells. It follows that 8H.8 does not produce cross-binding of mast cell-bound IgE. This finding is confirmed by fixation studies with a recombinant receptor described in this report.

Competitive trials are conducted between a antibody, 8H.8 or 15A.2, and soluble exon 3 to identify the level of inhibition of the binding of these antibodies on the Native IgE purified by affinity and immobilized on the phase solid of an ELISA test. The results of this study are reproduce in figure 3. Figure 3 shows that the increase of the concentration of recombinant exon 3 inhibits the fixation of 8H.8 monoclonal antibody, but does not inhibit antibody 15A.2.

The capacity of these is also examined monoclonal antibodies for inhibiting IgE binding on the solid phase recombinant IgE receptor assay ELISA

Competitive tests are also carried out with the 8H.8 antibody and similar assays with 15A.2, in which the antibody is in competition with canine IgE purified by affinity and that is in solution and the solid phase IgE of the ELISA test. The data of these tests are represented in the figure Four.

These studies suggest that 15A.2 has a great affinity with soluble native canine IgE because, when the concentration of soluble IgE increases, the 15A.2 binding on immobilized immunoglobulin, indicating that 15A.2 is now fixed on soluble IgE. Instead, it is observed that the 8H.8 antibody has a much lower affinity with soluble IgE, because increasing concentrations of IgE soluble do not inhibit the ability of 8H.8 to be fixed on IgE immobilized The data therefore suggests that soluble IgE is not effective to inhibit 8H.8 binding on immobilized IgE On a solid phase. Therefore, the affinity of 8H.8 with the Soluble canine IgE is less than the affinity of this antibody monoclonal with the solid phase IgE.

These studies also suggest that 8H.8 has poor affinity with soluble native canine IgE, because the increasing concentrations of soluble IgE do not inhibit the ability of 8H.8 to be fixed on the immobilized IgE. Unlike that, it is observed that antibody 15A.2 has a much higher affinity with soluble IgE, because as the concentration of soluble IgE significantly decreases the fixation of 15A.2 on immobilized immunoglobulin, thus indicating that the 15A.2 is currently fixed on soluble IgE. Thus, the data suggest that soluble IgE is not effective in inhibiting 8H.8 fixation on the immobilized IgE on the solid phase. Therefore, the affinity of 8H.8 with soluble canine IgE is less than the affinity of this monoclonal antibody with the IgE of the solid phase

To better delimit the finding that 15A.2 it has greater affinity with soluble native IgE and 8H.8 has less affinity with soluble native IgE studies are conducted in which 8H.8 and similar studies with 15A.2 are used to determine to what extent each antibody inhibits the binding of soluble IgE on a solid phase of recombinant IgE receptor. As a means of detection is used in the studies represented in figure 5 is employs an antibody labeled D9. It is known that the labeled antibody D9 is fixed on the IgE when it is bound to the receptor. In the studies represented in figure 5, native IgE is present at a concentration of 2.5 micrograms per milliliter. As it indicated in figure 5, 15A.2 is effective in inhibiting fixation of native IgE on the recombinant IgE receptor in very low antibody concentrations, of the order of 190 nanograms per milliliter Unlike that, 3,125 nanograms are required per milliliter of 8H.8 to inhibit native IgE fixation on the recombinant IgE receptor. Therefore, since it requires more 8H.8 than 15A.2 to inhibit IgE fixation native on the recombinant receptor, it follows that 8H.8 has lower affinity with soluble IgE. It is revealed therefore that when a sufficient concentration of the antibody is provided 8H.8 or 15A.2, then the antibodies damage or reduce the fixation of soluble IgE on the IgE receptor.

It is also observed, by the data represented in Figure 2, that the avidity of 8H.8 for fixing native canine IgE It increases greatly when IgE is immobilized. These studies suggest that 8H.8 has a low affinity with IgE native soluble canine, but when said IgE is immobilized on a surface, or when it has been expressed on the surface of a memory B cell, then greatly increases the avidity of 8H.8 to look at the native canine IgE.

Therefore, in view of these findings, has hypothesized that the region within which recognizes native IgE by 8H.8 could be partially hidden, particularly when IgE is in the serum. A amino acid sequence of IgE that is partially sequestered cannot be easily exposed to the native canine immune system or its normal protection mechanisms. Also, since exon 3 of full length does not lead to the generation of antibodies that have specificity similar to that of 8H.8, the length sequence complete may contain suppressor peptides capable of regulating the unsubscribe or delete an answer auto-anti-IgE, which is specific against the epitope recognized by the 8H.8 antibody. Any of the two mechanisms can serve to prevent the production of a autoimmune response to auto-antigen.

The implications of these findings are that the 8H.8 antibody, when used in allergy therapy in dogs, will be preferentially fixed on the IgE of the B cells of memory, before doing so with the IgE in solution. In addition, 8H.8 it has a low binding affinity with the IgE that is fixed by the mast cell Fc receptor.

To map the epitope bound by the antibody 8H.8 phage display technology is used (phage display technology). A preferred method of carrying out the phage display technology is associated with the use of the kit called "Ph. D. Phage Display Peptide Library Kit" of the New England Biolabs, Beverly, Mass. It has been found that a 7 amino acid peptide (Thr-Leu-Leu-Glu-Tyr-Arg-Met) inhibits competitive binding of monoclonal antibody 8H.8 both on the native canine IgE as on the recombinant. This sequence contains 6 common amino acids in the form and / or spacing with the C-terminated region of exon 3. A peptide of 11 amino acids synthesized from this region (Gly-Met-Asn-Leu-Thr-Trp-Tyr-Arg-Glu-Ser-Lys), denominated E3a.5, it also inhibits the competitive fixation of 8H.8 monoclonal antibody on recombinant or native IgE.

It is believed that antibody responses with specificity, for example those of 8H.8, do not take place so natural, even after the events that induce auto-anti-IgE responses to other epitopes, because epitope 8H.8 is available only partially for recognition. However, the hypothesis that antibodies to an epitope of type 8H.8 generated by peptide immunization according to the invention produce fixation on the partially available epitope, such as the produces the 8H.8 monoclonal antibody.

It is also hypothesized that the region of the native IgE that is recognized by 15A.2 can serve as immunogen to induce responses auto-anti-IgE and can generate antibodies capable of binding on IgE + B cells and regulating in decreasing sense the synthesis of IgE. The implications of These findings are that the 15A.2 antibody, when used for The therapeutic treatment of canine allergy will prevent IgE fixation on mast cells and will potentially affect the synthesis of IgE in B cells.

To map the epitope bound by the antibody 15A.2 phage display technology is used (phage display technology). As indicated before, a method preferred to carry out phage display technology It is associated with the use of the kit called "Ph. D. Phage Display Peptide Library Kit "from New England Biolabs, Beverly, Mass. The amino acid sequences of the PhDc7c library, which are fixed by the monoclonal antibody 15A.2. These sequences are aligned with the sequence of canine IgE protein. Figure 7 shows the alignment of epitope 15A.2 of seven different mammals: dog, Man, green monkey, cat, pig, mouse and horse.

Figure 8 shows the capacity of 15A.2 mimotope peptides that have different phage inhibits that canine IgE fix monoclonal antibody 15A.2 on the phase solid. 466 µL of biotinylated antibody 15A.2 (10 µg / ml) with 466 µl phage from a fresh crop of one night They are introduced into each hole of the plate 100 µl microtiter of the mixture. It is left at rest for 2.5 hours for antibodies to bind to phages. TO The holes are then coated with streptavidin. They wash the plates three times with standard wash buffer to remove the Loose set material. A series of dilutions is prepared of canine IgE, starting from the concentration of 1 \ mug / \ mul of PBS, 0.1% Tween. 100 µl of the IgE dilution is added to each hole and let it set for 10 minutes. The competition reaction by washing the plate five times with wash solution The plate with antibody is revealed below monoclonal D9 attached to HRPO, which is fixed on domain 4 of the IgE, to visualize the IgE of the solid phase. Signal loss indicates that the phage has successfully repelled the competition of the IgE canine Table 1 shows the concentrations of the reagents at the various points on the x axis of Figure 8.

TABLE 1

2

Using the display library of phage PhD12 of the New England Biolabs company shows that a 12 amino acid peptide (SEQ ID NO: 11, Val-Thr-Leu-Cys-Pro-Asn-Pro-His-Ile-Pro-Met-Cys) inhibits competitive binding of monoclonal antibody 15A.2 both on the native canine IgE as on the recombinant. This sequence contains 4 common amino acids in the form and / or spacing with the N-terminated region of exon 3.

The capacity of the monoclonal antibody 15A.2 to fix a synthetic peptide. Be synthesizes the peptide of SEQ ID NO: 12, Ser-Val-Thr-Leu-Cys-Pro-Asn-Pro-His-Ile-Pro-Met-Cys-Gly-Gly-Gly-Lys and biotinylates on the epsilon carbon of the rest Lys. This sequence corresponds to the isolated M13-48. It's about peptide so as to promote the reduction and cyclization of cysteines to form a cyclic peptide. A series of dilutions of the peptide and fixed to microtiter plates coated with streptavidin (5 µg of streptavidin per hole). The peptide bound with the monoclonal antibody is detected 15A.2 conjugated with HRPO. The results are shown in figure 9 of this essay. This phase-specific 15A.2 mimotopo peptide solid is fixed on monoclonal antibody 15A.2 in a manner which is concentration dependent.

In Figures 10A and 10B the ability of the mimotope peptide of 15A.2 to prevent fixation of the monoclonal antibody 15A.2 on the canine IgE of the solid phase. Plates are coated with canine IgE in a concentration of 1 \ mug / ml. A series of dilutions of the synthetic peptide is added to the monoclonal antibody 15A.2 conjugated with HRPO (final concentration: 1 µl / ml) and allowed to stand for two hours. 100 µL of the 15A.2 / peptide mixture is added to the pits and leave at rest to join for 1 hour. The reaction by washing the plates, six times, with wash buffer. The plates with HRPO substrate are revealed, the reactions with a mixture to that effect and densities are measured Optics (OD) of the plates with a plate reader.

Apart from active immunization with antibody 15A.2 or 8H.8 to induce the production of auto-anti-IgE " in vivo ", this invention also relates to a specific binding molecule that specifically binds a ligand of the type set by 15A.2 or 8H.8, as well as the therapeutic or prophylactic use thereof. The invention further relates to a specific monoclonal antibody of a conservative variant of a sequence set by 15A.2, of a conservative variant of a sequence fixed by 8H.8 or of a native canine IgE sequence located at a distance up to 100 amino acids of the C 'terminal portion of exon 3. The specific binding molecules of the invention can be used in a method according to the invention for the treatment or prophylaxis of allergy symptoms, that is, for passive immunization.

Therefore, administration to a dog already is of an antibody according to this invention, for example 15A.2 or 8H.8, or of a peptide according to this invention, for example the peptide of 7, 11 or 12 amino acids, leads to a decrease in subsequent production of IgE. This can be done by example by fixing antibody 15A.2 or 8H.8 at memory B cells and by depriving the posterior IgE production. In the case of a peptide, its administration will lead to the production of specificity and effect antibodies comparable to those of 15A.2 or 8H.8.

Examples Example 1

The results of the fixation study in which the 8H.8 antibody and the antibody 15A.2 are tested separately to determine their ability to look at a solid phase that has the IgE immobilized affinity purified canine. These results indicate that 8H.8 and 15A.2 antibodies are fixed on native IgE when it is immobilized on a solid phase. The results of these studies suggest that the binding of each of these antibodies to IgE attached to the surface is of a similar affinity. To determine if the binding affinity of each antibody is, in fact, comparable, complementary studies are carried out.

The results of the study in which the 8H.8 antibody conjugated to a signal residue it is reacted in various concentrations with solid phases that they have various peptides immobilized on them. The data represented by (\ blacklozenge) reflect a solid phase have the canine IgE immobilized on it; the data represented by (\ blacksquare) reflect a solid phase that it has the E3a.5 immobilized on it; the data represented with (\ Pi) reflect a solid phase that has an extended peptide with E3a.5 immobilized on it; the data represented by (X) reflect the data of a solid phase that has a peptide encoded with E3a.5 immobilized on it; the data represented by a (*) reflect a solid phase that has a seven amino acid peptide, identified with the technology of phage display, which is fixed on the SH8, immobilized on she. It is therefore observed that 8H.8 is fixed on each phase solid, except on the solid phase of the encoded peptide with E3a.5 immobilized on it.

It is known that the 14K2 monoclonal antibody is fixed exclusively on exon 4 of canine IgE. It reacts this antibody with various solid phases that have various peptides immobilized on them. The data represented by (\ blacklozenge) reflect a solid phase that has canine IgE immobilized on her; the data represented by (\ blacksquare) reflect a solid phase that has E3a.5 immobilized on her; the data represented with (\ Pi) they reflect a solid phase that has an extended peptide with E3a.5 immobilized on her; the data represented by (X) reflect the data of a solid phase that has a peptide encoded with E3a.5 immobilized on it; the data represented by a (*) reflect a solid phase that has a seven amino acid peptide, identified with the technology of phage display, fixed on 8H.8, frozen about her. It is therefore observed that the 14K2 is fixed only on a solid phase, which has the complete canine IgE molecule immobilized on her. These data are represented in the figure 13.

It is believed that monoclonal antibody 15A.2 is fixed only on exon 3 of canine IgE. Figure 14 shows represent data from studies that evaluate the fixation of 15A.2 on various peptides. The data represented by (\ blacklozenge) reflect a solid phase that has canine IgE immobilized on her; the data represented by (\ blacksquare) reflect a solid phase that has E3a.5 immobilized on her; the data represented with (\ Pi) they reflect a solid phase that has an extended peptide with E3a.5 immobilized on her; the data represented by (X) reflect the data of a solid phase that has a peptide encoded with E3a.5 immobilized on it; the data represented by a (*) reflect a solid phase that has a seven amino acid peptide, identified with the technology of phage display, fixed on 8H.8, frozen about her. It is therefore observed that 15A.2 is fixed only on a solid phase, which has the canine IgE molecule complete immobilized on her. These data indicate that 15A.2 it is not fixed on the same epitope, on which 8H.8 is fixed.

Figure 15 shows the data of the studies comparing 8H.8 or antibody binding polyclonal cultured to become recombinant human IgE, on the E3a.5 peptide or on the E3a.5 peptide with a substitution of amino acid that makes it more analog of a sequence of human genome that encodes human IgE, called peptide S3a.5. In Figure 15, the data represented by (\ blacklozenge) they reflect the 3a.5 substituted and compared with the 8H.8; the data represented by (\ blacksquare) reflect peptide 3a.5 substituted and Re-Hu IgE; the data represented with (? Pi) they reflect an E3a.5 and an 8H.8 peptide and the data represented by (X) reflect peptide E3a.5 and the Re-Hu polyclonal IgE. These data indicate that only 8H.8 is fixed on E3a.5 in the solid phase, none of the remaining combinations have such fixation.

Figure 16 shows the data of studies comparing 8H.8 binding on various peptides immobilized on a solid phase. The data represented by (\ blacklozenge) they reflect a solid phase that has the IgE canine immobilized on it; the data represented by (\ blacksquare) reflect a solid phase that has E3a.5 spread immobilized on her; the data represented with (\ Pi) reflect a solid phase that has human IgE immobilized on her; the data represented by (X) reflect the data of a solid phase that has an 8H.8 peptide. immobilized on her. According to these data, it is observed that 8H.8 it is fixed on canine IgE, extended E3a.5 peptide or peptide of seven amino acids identified by the technology of phage display as 8H.8 fixation epitope, when each of these peptides is immobilized on a phase solid. It is also observed that 8H.8 is not fixed on IgE human

Figure 17 shows the data of studies comparing the capacity of polyclonal antibodies anti-human IgE to look at solid phases that have various peptides immobilized on them. The data represented by (\ blacklozenge) reflect a solid phase which has canine IgE immobilized on it; the data represented by (\ blacksquare) reflect a solid phase that it has the extended E3a.5 immobilized on it; the data represented with (\ Pi) reflect a solid phase that has the IgE human immobilized on her; the data represented by (X) reflect the data of a solid phase that has an 8H.8 peptide. immobilized on her. According to these data, it is observed that polyclonal anti-human IgE antibody is fixed on human IgE when immobilized on a solid phase. It is also observed that the polyclonal antiserum has a fixation limited on the E3a.5 peptide extended when antibodies polyclonal are present in very high concentrations; this It is believed to be a fixation device due to concentration high antibody Therefore, it is noted that the antibodies of human IgE do not specifically target canine IgE or on the peptides according to this invention.

Example 2

Table 2 shows the sequences of epitope amino acids fixed by 8H.8, the sequence of 11 amino acids used in immunization studies presented in this description as well as the analogous sequences of feline IgE and human

A substituted canine sequence is also prepared which has an amino acid substitution that makes the peptide more similar to the human peptide.

TABLE 2

3

Each of the peptides in Table 2 is placed on a solid phase and studies are conducted to identify if the 8H.8 is fixed on the surface-fixed peptide. Be find that 8H.8 is fixed to a surface that has the peptide mimotope on it and on a surface that has the 11 peptide amino acids fixed on it. It is noted that 8H.8 is not fixed on the human peptide, the feline peptide or the canine sequence which has an amino acid substitution that makes it more similar to the human sequence, when any one of these Peptides are fixed on the surface.

Conclusions

Unless otherwise indicated, all technical and scientific terms used in this description they have the same meaning that is usually attributed by part of the experts in the field, to which it belongs this invention. Although methods and materials can be used similar or equivalent to those described herein for the implementation or for the verification of the invention, the Preferred methods and materials are now described.

Claims (31)

1. A specific binding protein, chosen between the group formed by a monoclonal antibody, an antibody polyclonal, a fragment of monoclonal antibody that is fixed on an antigen, a fragment of a polyclonal antibody that binds on an antigen, a hybrid antibody and a chain antibody simple, which is specifically set on free native canine IgE or bound to B cells by fixing on a sequence of amino acids consisting of: (SEQ ID NO: 1) leucine-white-white-tyrosine-arginine; (SEQ ID NO: 2) tyrosine-arginine-white-white-leucine; (SEQ ID NO: 3) leucine-white-white-tyrosine-arginine-white-white-leucine; (SEQ ID NO: 4) threonine-leucine-leucine-acid glutamic-tyrosine-arginine-methionine; (SEQ ID NO: 5) glycine-methionine-asparagine-leucine-threonine-tryptophan-tyrosine-arginine-acid glutamic-serine-lysine; (SEQ ID NO: 6) cysteine-white-white-proline-histidine-white-white-white-cysteine; (SEQ ID NO: 7) serine-valine-threonine-leucine-cysteine-proline-asparagine-proline-histidine-isoleucine-proline-methionine-cysteine-glycine-glycine-glycine; (SEQ ID NO: 8) serine-alanine-cysteine-proline-asparagine-proline-histidine-asparagine-proline-tyrosine-cysteine-glycine-glycine-glycine; (SEQ ID NO: 9) cysteine-white-proline-histidine-white-proline-white-white-cysteine; (SEQ ID NO: 10) serine-alanine-cysteine-histidine-proline-histidine-leucine-proline-lysine-serine-cysteine-glycine-glycine-glycine; (SEQ ID NO: 11) valine-threonine-leucine-cysteine-proline-asparagine-proline-histidine-isoleucine-proline-methionine-cysteine; or (SEQ ID NO: 12) serine-valine-threonine-leucine-cysteine-proline-asparagine-proline-histidine-isoleucine-proline-methionine-cysteine-glycine-glycine-glycine; where white is any amino acid and that is not fixed on IgE when IgE is bound to an existing receiver in a mast cell 2. The specific binding protein of the claim 1, wherein the IgE fixed on cell B is the IgE expressed on the surface of a canine B cell. 3. The specific binding protein of the claim 1 or 2, which is specifically fixed on a peptide isolated and purified containing a leucine positioned two peptide bonds beyond the pair tyrosine-arginine. 4. The specific binding protein of the claim 3, which is specifically fixed on a peptide which It has SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. 5. The specific binding protein of the claim 4, wherein at least one blank is an amino acid It has an aromatic ring. 6. The specific binding protein of a any one of claims 1 to 5, which is a antibody. 7. The antibody of claim 6, which is a monoclonal antibody. 8. The antibody of claim 6, which is presents as an isolated and purified peptide that has a group leucine positioned two peptide bonds beyond the pair tyrosine-arginine and said peptide contains from 5 to 71 amino acids. 9. The antibody of claim 8, which is presents in the form of an isolated and purified peptide that contains a amino acid sequence, which consists of SEQ ID NO: 4 or a conservative variant of it. 10. The antibody of claim 8, which is presents in the form of an isolated and purified peptide that contains a amino acid sequence consisting of SEQ ID NO: 5 or a conservative variant of it. 11. The specific binding protein according to a any one of claims 1 to 10, which is presented in multiple antigenic peptide form consisting of multiple copies of an isolated and purified peptide containing a leucine group positioned two peptide bonds beyond the pair tyrosine-arginine. 12. The specific binding protein of the claim 11, said protein contains from 5 to 71 amino acids. 13. The specific binding protein of a any one of claims 1 to 12, which is presented in particle form of recombinant plant virus, which contains at least one copy of an isolated and purified peptide containing a leucine residue positioned two peptide bonds beyond the pair tyrosine-arginine. 14. The specific binding protein of the claim 13, said peptide contains from 5 to 71 amino acids. 15. The specific binding protein of a any one of claims 1 to 14, which is fixed specifically on a defined epitope. 16. The specific binding protein of the claim 15, which is a recombinant binding molecule. 17. The specific binding protein of the claim 1, wherein the specific binding protein is a monoclonal antibody derived from mice immunized with exon 3a of the canine IgE molecule, which is specifically fixed on free IgE or IgE bound to B cells and not fixed on IgE when IgE is bound to the IgE receptor. 18. The specific binding protein of the claim 1, wherein the specific binding protein is a monoclonal antibody derived from mice immunized with the affinity purified canine IgE molecule, which binds specifically to free IgE or IgE bound to B cells and that is not fixed on the IgE when the IgE is bound to the receptor of IgE 19. The specific binding protein of a any one of claims 1 to 3, which is fixed specifically on an isolated and purified peptide containing SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 12. 20. The specific binding protein of a any one of claims 1 to 3, which is fixed specifically on an isolated and purified peptide containing SEQ ID NO: 13 (cysteine-proline-asparagine-proline-histidine-isoleucine-proline-methionine-cysteine) or SEQ ID NO: 14 (cysteine-proline-asparagine-proline-histidine-asparagine-proline-tyrosine-cysteine). 21. The specific binding protein of the claim 20, wherein the peptide contains the sequence serine-alanine-cysteine-proline-asparagine-proline-histidine-asparagine-proline-tyrosine-cysteine-glycine-glycine-glycine. 22. The specific binding protein of a any one of claims 19 to 21, which is a antibody. 23. The antibody of claim 22, which is a monoclonal antibody. 24. The antibody of claim 22, which is presents in the form of isolated and purified peptide, provided with the SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 12 or a variant Conservative of them. 25. The antibody of claim 22, which is presents in the form of isolated and purified peptide, provided with the SEQ ID NO: 13 or SEQ ID NO: 14 or a conservative variant of the same. 26. The antibody of claim 25, in the that said peptide contains the sequence serine-alanine-cysteine-proline-asparagine-proline-histidine-asparagine-proline-tyrosine-cysteine-glycine-glycine-glycine. 27. The specific binding protein according to a any one of claims 19 to 26, which is fixed specifically on a defined epitope. 28. The specific binding protein of the claim 27, which is a recombinant binding molecule. 29. The use of a specific binding protein according to any one of claims 1 to 28 for the manufacture of a pharmaceutical composition intended for treatment or prophylaxis of canine allergy. 30. The use of claim 29, wherein the Pharmaceutical composition also contains a diluent. 31. The use of claim 29 or 30 for the manufacture of a pharmaceutical composition kit, said kit contains a first dose of said specific binding protein and a second dose of said specific binding protein.